A C-terminal CaaX motif, where C is cysteine, the a's are aliphatic amino acids, and X can be any of a number of amino acids, targets a variety of eukaryotic proteins to a series of post-translational modifications important for their localization and function (Zhang and Casey, Annu. Rev. Biochem. 65:241-269 (1996), Kloog and Cox, Semin. Cancer Biol. 14:253-261 (2004)). This processing is initiated by the covalent attachment of a 15-carbon farnesyl or a 20-carbon geranylgeranyl lipid to the cysteine of the CaaX motif, a reaction catalyzed by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I) (Casey and Seabra, J. Biol. Chem. 271:5289-5292 (1996)). Following prenylation, the C-terminal three amino acids (i.e., the -aaX) are removed by a specific endoprotease termed Rce1 (Boyartchuk et al, Science 275:1796-1800 (1997), Otto et al, J. Biol. Chem. 274:8379-8382 (1999)) and the now C-terminal prenylcysteine moiety is methylated by the enzyme isoprenylcysteine carboxyl methyltransferase (Icmt) (Clarke et al, Proc. Natl. Acad. Sci. USA 85:4643-4637 (1988), Hrycyna et al, EMBO J. 10:1699-1709 (1991), Dai et al, J. Biol. Chem. 273:15030-15034 (1998)). As polytopic membrane proteins that are localized to the endoplasmic reticulum, both Rce1 and Icmt are unusual in their respective classes (Young et al, The Enzymes 21:156-213 (2000)).
Proteins that terminate in a -CaaX motif regulate a number of cellular pathways important in oncogenesis. The best studied example is the central role of the Ras family of CaaX proteins in growth factor activation of the Raf/mitogen-activated protein kinase (MAPK) signaling cascade (Malumbres and Barbacid, Nat. Rev. Cancer 3:459-465 (2003), Shields et al, Trends Cell Biol. 10:147-154 (2000)). Constitutive activation of this pathway is transforming in a wide variety of cell types, and activating mutations in Ras have been found in almost 30% of all cancers, including 50% of colon cancers and up to 90% of pancreatic cancers (Bos, Cancer Res. 49:4682-4689 (1989)). In addition, many cancers contain alterations in elements upstream of Ras in signaling cascades, such as amplified expression or mutational activation of tyrosine kinases, and the resultant hyperactivation of Ras is thought to contribute to tumorigenesis in these cancers as well (Gschwind et al, Nat. Rev. Cancer 4:361-370 (2004), Schlessinger, Cell 103:211-225 (2000)). In addition to Ras, many other CaaX proteins have been implicated in oncogenesis and tumor progression, and these proteins also most likely require processing via the prenylation pathway for function (Kloog and Cox, Semin. Cancer Biol. 14:253-261 (2004), Doll et al, Curr. Opin. Drug. Discov. Devel. 7:478-486 (2004)).
Both the membrane targeting and the transforming abilities of Ras require processing through the prenylation pathway (Hancock et al, Cell. 57:1167-1177 (1989), Kato et al, Proc. Natl. Acad. Sci. USA 89:6403-6407 (1992)). For this reason, the protein prenyltransferases, most notably FTase, have been targets of major drug discovery programs for the last decade (Gibbs et al, Cell 77:175-178 (1994), Karp et al, Curr. Opin. Oncol. 13:470-476 (2001)). Presently, several FTase inhibitors (FTIs) are being evaluated in clinical trials (Doll et al, Curr. Opin. Drug. Discov. Devel. 7:478-486 (2004), Karp et al, Curr. Opin. Oncol. 13:470-476 (2001)). While these experimental agents have shown significant activity in a number of clinical trials, the overall response rates in patients have been less than initially hoped. One possible explanation for this lack of efficacy is the process of alternate prenylation that allows some FTase substrates to be modified by GGTase-I when FTase activity is limiting (James et al, J. Biol. Chem. 270:6221-6226 (1995), Whyte et al, J. Biol. Chem. 272:14459-14464 (1997), Sebti and Der, Nat. Rev. Cancer 3:945-951 (2003)). Recent studies using genetic disruption of Icmt have demonstrated that Ras proteins, including K-Ras, are significantly mislocalized and tumorigenesis is markedly impaired in cells that lack Icmt (Bergo et al, J. Clin. Invest. 113:539-550 (2004), Bergo et al, J. Biol. Chem. 275:17605-17610 (2000)). Following this discovery, CaaX protein methylation has gained attention as a new target in oncogenesis (Clarke and Tamanoi, J. Clin. Invest. 113:513-515 (2004)).
With emerging evidence for the importance of Icmt-catalyzed CaaX protein methylation in oncogenesis, there is a clear need for specific pharmacological agents to target this process. However, the only such agents available to date have been analogs of the substrate prenylcysteine or the product S-adenosylhomocysteine; all of these have been reported to have pleiotropic effects on cells (Chiang et al, FASEB J. 10:471-480 (1996), Ma et al, Biochemistry 33:5414-5420 (1994), Scheer and Gierschik, FEBS Lett. 319:110-114 (1993)). (See also Winter-Van and Casey, Nature Rev. Cancer 5:405-412 (2005).)
The present invention results, at least in part, from the discovery of a novel, indole-based small-molecule inhibitor of Icmt. Treatment of cancer cells with this compound, designated cysmethynil, results in a decrease in Ras carboxylmethylation, mislocalization of Ras, and impaired signaling through Ras pathways. The invention provides methods of treating diseases or disorders dependent on activity of Icmt substrates, and, more specifically, diseases or disorders associated with aberrant activity of Icmt substrates (e.g., cancer) using this and other Icmt inhibitors.